This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Post-translational modifications can diversify the functional properties of gene products and is a widely-utilized strategy in biology. Diphthamide [2,3-carboxyamido-3- (trimethylammonio)propyl histidine] is a uniquely-modified histidine residue found conserved in translation elongation factor 2 from both eukaryotes and archaebacteria. The function of diphthamide is still the subject of research. For example, yeast mutants lacking the modification have been identified with uninhibited growth and viability (Chen et al. Mol. Cell Biol. 1985). However, the diphthamide residue appears to have function in preventing -1 frameshifting during protein synthesis (Gomez-Lorenzo et al. EMBO J. 2000) and is also the ADP-ribosylation target of diphtheria toxin as well as Pseudomonas aeruginosa exotoxin A. The biosynthesis of diphthamide is a complex process that involves 6 proteins in eukaryotes, dph1-5 and an as-yet unidentified enzyme that performs a final amidation step. The first step involves the transfer of the 3-amino-3-carboxypropyl group from S-adenosylmethionine (SAM) and involves the enzymes dph1-4. Dph-5 catalyzes trimethylation of the amino group to form diphthine, the substrate for the final amidation step. Interestingly, only 2 gene products have been identified in archaebacteria to be involved in diphthamide synthesis, dph-2 and dph-5. Structural studies have been undertaken on archaebacterial dph-2 in order to provide insight into the diphthamide biosynthetic pathway.